KSHV is an oncogenic herpesvirus, which is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and aggressive forms of multicentric Castleman's disease. Importantly, the establishment of viral latency in the host is thought to be one of the major driving forces of KSHV pathogenesis. During latency the latent genes (LANA, v-Cyclin, K13, v-miRNAs and K12) are constitutively expressed, while the lytic genes are repressed. The latent viral products have growth transforming and cell cycle-deregulating properties that are involved in the development of KSHV-associated cancers. Despite the importance of viral latency in KSHV pathogenesis, the molecular mechanism of the establishment of KSHV latency is still poorly understood. Thus, understanding how KSHV establishes latency is the primary goal of our proposal. We have recently demonstrated that following de novo infection, the KSHV genome first acquires a transcriptionally permissive chromatin allowing the expression of lytic genes, which is followed by the binding of the cellular Polycomb Repressive Complexes (PRC1 and PRC2) to the viral episome that results in the inhibition of lytic genes leading to the establishment of viral latency. Our preliminary results indicate that the latent KSHV protein called latency-associated nuclear antigen (LANA) is required for the recruitment of the H3K27me3 histone methyltransferase EZH2 of PRC2 to the KSHV genome during de novo infection. Based on our preliminary data, we propose to dissect the molecular mechanism of how LANA recruits the Polycomb proteins onto the KSHV genome, which can serve as the primary means of the repression of lytic genes following de novo infection that ultimately leads to the establishment of viral latency. In Aim 1, we will investigae the mechanism of LANA-mediated EZH2 recruitment to the KSHV genome by using LANA mutant KSHV clones for de novo infection, and test the recruitment of EZH2 to lytic promoters. In Aim 2, we will test the functional role of LANA-EZH2 interaction in viral gene expression during de novo infection by using the innovative NanoString's nCounter technology, which utilizes a digitally color-coded barcode method and single molecule imaging to detect KSHV mRNAs in a single reaction.